Grupa T. Smital – Molekularna ekotoksikologija

Cilj istraživanja– temeljna ekotoksikološka istraživanja usmjerena na razumijevanje:

●  Molekularne osnove i uloge staničnih detoksifikacijskih mehanizama u organizmima vodenih ekosistema

●  Interakcije staničnih detoksifikacijskih mehanizama sa klasičnim i novim okolišnim zagađivalima

Krajnji cilj gore navedenih temeljnih istraživanja je vrednovanje uloge staničnih detoksifikacijskih mehanizama kao ključnih sastavnica tzv. ADMETox obilježja okolišnih zagađivala. Rezultati naših istraživanja doprinijet će i poboljšanju i/ili razvoju ekotoksikoloških molekularnih biomarkera tzv. ‘ranog upozorenja’ te razvoju tzv.high-throughput alata za evaluaciju pojedinačnih okolišnih zagađivala, kao i evaluaciju kompleksnih okolišnih mješavina zagađivala.

1.Razumijevanje ekotoksikološkog značaja:

a) Proteina eksportera: ABC (ATP Binding Cassette) i MATE (Multidrug and Toxin extrusion) transportnih proteina (effluxtransportera)

b) Proteina unosnika: SLC (Solute Carriers) - SLC21/OATP and SLC21 (OAT, OCT) transportnih proteina (uptaketransportera)

c) GST (Glutathione S-transferase) enzima: faza 2 staničnog ADME (Administration, Distribution, Metabolism, Elimination)

2.Razvoj tzv. Effects-Directed Analyses(EDA)pristupa u svrhu identifikacije štetnih kemijskih zagađivala u okolišu.

2017- present – Group leader, Tenure-track Research Associate

Grant holder

 1. 2020-2024 ‘Structural characterization of factors involved in DNA-protein crosslink repair‘ - Slovenian-Croatian Bilateral Project (IPS-2020-01) (1.5 million HRK), Institute Ruder Boskovic, Croatia and National Chemistry Institute, Slovenia (300,000 EUR), total project value 500,000 EUR) . Collaborators: dr.sc Marjetka Podobnik (SLO) and dr.sc. Nives Ivić (IRB, HR)

 2. 2018-2023 ‘Deciphering DNA-Protein Crosslink Repair in vivo using CRISPR/Cas9 genome editing in zebrafish model ’Croatian National Science Foundation Installation project grant (1.86 million HRK), Institute Ruder Boskovic, Croatia.

Popovic lab webpage available at:

https://martafry.wixsite.com/popoviclab

Members:

Group leader, Senior Research Associate: Marta Popovic, PhD

Tenure-track Research Associate: Cecile Otten, PhD

Postdoc: Ivan Anticevic, PhD

PhD student: Marin Kutnjak, MSc molecular biology

Project title: Structural characterization of factors involved in DNA-protein crosslink repair (2020-2023)

DNA-protein crosslinks (DPCs) are severe DNA lesions which occur when a protein becomes irreversibly covalently linked to DNA. They have adverse effects on the organismal level including cancer, premature aging and neurodegenerative diseases. Due to their bulky nature, DPCs impair all DNA transactions (replication, transcription and repair) and DPC repair is therefore an essential cellular pathway. Despite the fact that DPCs are frequently occurring in the nucleus and cause severe damage on a cellular level, not much is known about their repair mechanisms at a molecular level. Our structural studies aim to solve near-atomic details of the SPRTN-dependent DPC repair complex, the SPRTN:p97 complex and a novel DPC factor, ACRC, for the first time. Obtained knowledge will be fundamentally important for a deeper understanding of the repair pathway and thus for the developing research on p97 and SPRTN inhibitors for the purpose of targeted clinical therapies in cancerogenesis and aging. In terms of methodology, proposed studies will offer novel approaches for investigating DNA repair complexes and disordered protein regions using cryo-EM. Cryo-EM (Cryogenic electron microscopy) is a powerful approach to solve structures of large proteins and protein complexes. The importance of the approach was further emphasized in 2017., when the Nobel Prize in Chemistry was awarded to J. Dubochet, J. Frank, R. Henderson and "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution." (https://www.nature.com/news/cryo-electron-microscopy-wins-chemistry-nobel-1.22738;https://www.chemistryworld.com/news/explainer-what-is-cryo-electron-microscopy/3008091.article#/)

Project title: Deciphering DNA-Protein Crosslink Repair in vivo using zebrafish model (2018-2023)

Unakrsna veza DNA-protein (eng.DNA-protein crosslink;DPC) je tip DNA oštećenja koje nastaje kao posljedica ireverzibilne kovalentne veze između DNA i proteina u prisutnosti endogenog ili egzogenog DPC inducirajućeg agensa. Endogeni DPC induceri su nusprodukti staničnog metabolizma poput oksidativnih radikala, aldehida i promjene u DNA strukturi, dok su primjeri egzogenih inducera ultraljubičasto i ionizirajuće zračenje te različiti kemijski agensi. DPC-i su česta DNA oštećenja i predstavljaju fizičku prepreku za sve DNA transakcije: replikaciju, transkripciju, rekombinaciju i popravak. Nemogućnost popravka DPC-ova uzrokuje genomsku nestabilnost i teške posljedice za organizam, uključujući preuranjeno starenje, neurodegenreaciju i pojavu tumora. Unatoč visokoj učestalosti pojave DPC-a, malo se zna o mehanizmu njihova popravka, jer se sve donedavno DPC popravak nije smatrao zasebnim mehanizom popravka DNA. Tijekom 2014. i 2016. god. nekoliko grupa je identificiralo nove stanične enzime, proteaze Wss1 i SPRTN, koje proteolizom DPC-a iniciraju njihov popravak. Otkriće proteolizno-ovisnog popravka DPC-a dovelo je do prepoznavanja DPC popravka kao zasebnog mehanizma popravka DNA oštećenja. Međutim, trenutno nije poznata regulacija cijelog mehanizma, niti je poznato koji drugi faktori sudjeluju u popravku, dok gotovo ništa nije poznato o mehanizmu DPC popravkain vivo. Stoga je glavni cilj predloženog projekta otkriti regulaciju puta popravka DPC-ainvivokoristeći ribu zebricu (Danio rerio) kao istraživački model. Koristit ćemo CRISPR/Cas9 gensku manipulaciju kako bi utišali ili mutirali gen(e) od interesa, a za koji pretpostavljamo da su uključeni u DPC popravak. Uloga pojedinih proteina (i njihovih kombinacija) u popravku DPC-a biti će analizirana nakon izolacije DPC-a iz transgeničnih embrija i odraslih jedinki zebrice. Također ćemo konstruirati GFP reporter test u staničnim linijama i transgenim ribama koji će omogućiti kvantifikaciju učinkovitosti DPC popravka in vitro i in vivo.

More information about the project: https://www.irb.hr/eng/Research/Divisions/Division-for-Marine-and-Environmental-Research/Laboratory-for-molecular-ecotoxicology/DNAPRO-Deciphering-DNA-Protein-Crosslink-Repair-in-vivo-using-zebrafish-model

Open positions

A 3-year postdoctoral position is available in Popovic lab. Applicants should have a PhD in cell biology, molecular biology, biochemistry or a related field. The position will be open in February, 2025 and the start date is negotiable.

The postdoc will work on the mechanisms of DNA-protein crosslink repair and identification of the molecular causes of neurodegenerative diseases and carcinogenesis using stable human cell lines and the zebrafish animal model. Candidates with no prior experience in zebrafish work are welcome to apply (they will attend the zebrafish husbandry course in Karlsruhe, Germany after starting their work in the lab).

Interested candidates should contact the PI as soon as possible and send their CV for shortlisting of candidates (mpopovic@irb.hr).

A tenure-track Staff Scientist position is available in Popovic lab. The position is opened until filled and the start date is negotiable (within 2025 or 2026). Applicants should have a PhD in biochemistry, molecular biology or a related field and at least 4-5 years of international postdoctoral experience. The role of the Staff Scientist will be to support the research of the principal investigator by providing expertise in experimental techniques, helping to lead and manage experimental design, data collection and analysis, and mentoring of other lab members including pre- and post-doctoral trainees. Preference will be given to candidates with expertise in imaging (high-resolution light and fluorescence microscopy), sample preparation (e.g. cryosections of tissues), imaging data analysis, protein purifications using chromatography system (FPLC), biochemical methods for studying DNA (and RNA)-protein and protein-protein interactions and/or basics of X-ray crystallography. In addition, the applicant should be willing to work with the zebrafish animal model (husbandry).

Interested candidates should contact PI for additional information (mpopovic@irb.hr).